DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY.

An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3'-terminal phosphate monoester and 3'-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N(3) (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

Protein-aptamer binding studies using microchip affinity capillary electrophoresis.

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis.

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.

Myf5-/- :MyoD-/- amyogenic fetuses reveal the importance of early contraction and static loading by striated muscle in mouse skeletogenesis.

The mechanical loading of striated muscle is thought to play an important role in shaping bones and joints. Here, we examine skeletogenesis in late embryogenesis (embryonic day 18.5) in Myf5-/-:MyoD-/- fetuses completely lacking striated muscle. The phenotype includes enlarged and fused cervical vertebrae and postural anomalies, some viscerocranial anomalies, long bone truncation and fusion, absent deltoid tuberosity of the humerus, scapular and clavicular hypoplasia, cleft palate, and cleft sternum. In contrast, neurocranial bone development was essentially normal. While the magnitude of individual effects varied throughout the skeletal system, the results are consistent with skeletal development depending on functional muscles. Novel abnormalities in the amyogenic fetuses relative to less severely paralyzed phenotypes extend our understanding of skeletogenic dependence on embryonic muscle contraction and static loading.

The autofluorescence of plastic materials and chips measured under laser irradiation.

Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.

Arrested development in Xenopus laevis tadpoles: how size constrains metamorphosis.

Xenopus laevis tadpoles that arrest development and remain as larvae for several years sometimes occur spontaneously in laboratory populations. These tadpoles cease development at an early hindlimb stage, but continue to grow and develop into grossly deformed giants. Giant tadpoles lack thyroid glands, and differ in morphology and behaviour from normal larvae. They are negatively buoyant, typically with small and partially solidified lungs, and have greatly enlarged fat bodies. Giant tadpoles have mature gonads with eggs and sperm, whereas normal tadpoles of the same stage have undifferentiated gonads. Larval reproduction has never been reported in anurans, but gonadal development decoupled from metamorphosis brings these giants the closest of any anurans to being truly neotenic. We discuss behavioural and morphological factors that may hinder both reproduction in giant Xenopus larvae and the evolution of neoteny in anurans in general. Experimental treatment with exogenous thyroid hormone induces some, but not complete, metamorphic changes in these giants. The limbs and head progress through metamorphosis; however, all tadpoles die at the stage when the tail would normally be resorbed. The disproportionate growth of tissues and organs in giant tadpoles may preclude complete metamorphosis, even under exogenous thyroid hormone induction.

Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae.

Tadpoles that spontaneously arrest development and remain as larvae occur occasionally in Xenopus laevis populations. These non-metamorphosing tadpoles continue to grow, and they develop into grossly deformed giant individuals which come as close as any anurans to being truly neotenic. Giant X. laevis tadpoles that fail to metamorphose lack thyroid glands. In this study, the hypothesis that the tissues of these tadpoles nevertheless remain thyroid hormone sensitive was tested, by exposing isolated tadpole tail tips to exogenous thyroid hormone in tissue culture. The tail tips from giant tadpoles significantly shrank in response to the thyroid hormone treatment, showing that their tissue was still capable of metamorphosis. However, the amount of shrinkage was less than that observed in tail tissue from normal tadpoles. It was hypothesized that complete induction of metamorphosis may not be possible in the giant tadpoles due to a disproportionate growth and development of tissues and organs.